Bifunctional Role of Protein Tyrosine Kinases in Late
Preconditioning Against Myocardial Stunning
in Conscious Rabbits
Buddhadeb Dawn, Yu-Ting Xuan, Yumin Qiu, Hitoshi Takano, Xian-Liang Tang, Peipei Ping,
Supratim Banerjee, Michael Hill, Roberto Bolli
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Abstract—Although protein tyrosine kinases (PTKs) have been implicated in late preconditioning (PC) against infarction,
their role in late PC against stunning is unknown. Furthermore, it is unknown whether PTK signaling is necessary only
to trigger late PC on day 1 or also to mediate it on day 2. Thus, conscious rabbits underwent a sequence of six 4-minute
coronary occlusion/4-minute reperfusion cycles for 3 consecutive days (days 1, 2, and 3). In the control group (group
I, n57), the recovery of systolic wall thickening after the 6 occlusion/reperfusion cycles was markedly improved on days
2 and 3 compared with day 1, indicating the development of late PC against stunning. Administration of the PTK
inhibitor lavendustin-A (LD-A, 1 mg/kg IV) before the first occlusion on day 1 (group II, n57) completely prevented
the late PC effect against stunning on day 2. Late PC against stunning was also abrogated when LD-A was given before
the first occlusion on day 2 (group III, n57); however, in these rabbits, the late PC effect became apparent on day 3,
indicating that LD-A itself did not have any delayed deleterious actions on myocardial stunning. In group V (n55), the
sequence of 6 occlusion/reperfusion cycles resulted in a robust increase in the activity of inducible NO synthase (iNOS
[assessed as Ca21-independent L-citrulline formation]) and nitrite1nitrate (NOx) tissue levels 24 hours later (on day 2),
with no concomitant change in Ca21-dependent NO synthase (endothelial NO synthase and/or neuronal NO synthase)
activity. Similar results were obtained on day 3 (group VIII, n56), indicating sustained upregulation of iNOS.
Administration of LD-A either on day 1 (group VI, n55) or on day 2 (group VII, n56) abrogated the increase in iNOS
activity and NOx levels on day 2. LD-A had no effect on iNOS activity or NOx levels in the absence of PC (group X,
n55). This study demonstrates that in conscious rabbits, PTK activity is necessary not only to trigger late PC against
stunning on day 1 but also to mediate the protection on day 2. This investigation also provides the first direct evidence
that cardiac iNOS activity is upregulated during the late phase of ischemic PC in rabbits. Furthermore, the data indicate
that PTK signaling is essential for the augmentation of iNOS activity and that PTKs modulate this enzyme at two distinct
levels: at an early stage on day 1 and at a late stage on day 2. This bifunctional role of PTKs in late PC has broad
implications for the signaling mechanisms that underlie the response of the heart to ischemic stress and, possibly,
other stresses. (Circ Res. 1999;85:1154-1163.)
Key Words: myocardial stunning n protein tyrosine kinase n lavendustin A n inducible nitric oxide synthase
n ischemia/reperfusion injury
I
tection during subsequent ischemic insult(s) (reviewed in
Reference 8). Although reactive oxygen species9,10 and protein kinase C (PKC)11–14 are known to be involved in the
genesis of late PC, the exact signal transduction mechanism
whereby a brief ischemic stress leads to subsequent cardioprotection remains unclear. Elucidation of the cellular basis
of late PC is an essential step toward harnessing this cardioprotective phenomenon for therapeutic benefit.
Protein tyrosine kinases (PTKs), a diverse family of
enzymes that transfer phosphate from ATP to tyrosine residues on specific cellular proteins, are known to mediate a
schemic preconditioning (PC) confers myocardial protection in two temporally distinct phases: an early phase,
which develops immediately and lasts for 2 to 4 hours after
the ischemic stimulus, and a late phase, which begins after 12
to 24 hours and lasts for 3 to 4 days.1–3 Recent studies indicate
that NO plays a dual role (trigger and mediator) in late
PC.1,4 – 8 Specifically, the generation of NO associated with an
ischemic stress initiates a cascade of events that involves the
activation of several kinases and transcription factors and
culminates in the upregulation of the inducible NO synthase
(iNOS) gene; NO produced by iNOS then confers cardiopro-
Received June 22, 1999; accepted September 27, 1999.
From the Experimental Research Laboratory, Division of Cardiology, University of Louisville and Jewish Hospital Heart and Lung Institute,
Louisville, Ky.
This manuscript was sent to Stephen F. Vatner, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Correspondence to Roberto Bolli, MD, Division of Cardiology, University of Louisville, Louisville, KY 40292. E-mail [email protected]
© 1999 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
1154
Dawn et al
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wide variety of cellular responses.15–17 The nonreceptor PTKs
are specifically designed for signal transduction from cell
surface to intracellular enzymes and factors, usually by
protein-protein interactions.16 Recent evidence indicates that
PTKs play a role in the signaling mechanism underlying the
early18 –21 and the late22 phases of PC. Specifically, in isolated
rat hearts, inhibition of PTKs has been found to block the
PC-induced increase in the activity of phospholipase D
(PLD), PKC, and mitogen-activated protein kinase–activated
protein kinase 2 (MAPKAPK 2) as well as the cardioprotective effects of early PC,19 suggesting that the PTK-PLD-PKCMAPKAPK 2 signaling pathway plays a role in the early
phase of PC. Using isolated rabbit hearts, Baines et al20 have
demonstrated that the protection afforded by early PC against
myocardial infarction is abrogated by the PTK inhibitors
genistein and lavendustin A (LD-A). In addition, genistein
has been reported to block the development of late PC against
infarction in open-chest rabbits.22
Although these studies18 –22 implicate a PTK-dependent
pathway in the genesis of early and late PC, a number of
important issues remain to be addressed: First, while the
available evidence suggests an involvement of PTKs in late
PC against myocardial infarction,22 virtually nothing is
known regarding the role of PTKs in the genesis of late PC
against myocardial stunning. Myocardial stunning and infarction represent two very different types of injury, so that the
effects of PC on one cannot be extrapolated to the other. For
example, in dogs, the early phase of PC confers powerful
protection against myocardial infarction23,24 but fails to protect against the stunning induced by a 10- or 15-minute
coronary occlusion.25–27 Conversely, in conscious pigs, a
sequence of ten 2-minute coronary occlusions elicits a late PC
effect against stunning9,28,29 but not against infarction.30
These examples of a dissociation between the effects of PC
on stunning and infarction underscore the notion that, at least
under certain experimental conditions, different mechanisms
may be involved in the PC protection against reversible and
irreversible ischemic injury.25 Second, the precise role(s) of
PTKs in late PC has not been fully elucidated. In this regard,
it is important to distinguish the cellular mechanisms that
initiate the development of late PC immediately after the first
ischemic stress (day 1) from those that mediate cardioprotection 24 to 72 hours later (days 2 to 4). Although one previous
study22 examined the effect of genistein on the development
of late PC against infarction on day 1, the role of PTKs as
mediators of cardioprotection on days 2 to 4 has not been
explored. Thus, it is unknown whether PTK-dependent signaling is important only to trigger or also to mediate late PC.
Third, virtually nothing is known regarding the cellular
mechanisms by which PTKs contribute to late PC on day 1 or
day 2. Although several studies have implicated iNOS as the
mediator of late PC,5,7,8 direct evidence that iNOS activity is
augmented in the rabbit model of late PC is still lacking, and
the role of PTKs in iNOS modulation is unknown.
The present study was undertaken to address these issues.
We tested the hypothesis that PTKs play a dual role in the
pathophysiology of late PC against myocardial stunning, ie,
that they are essential not only for the initiation of this
phenomenon on day 1 but also for the manifestation of
Tyrosine Kinases and Late Preconditioning
1155
cardioprotection on day 2. We further hypothesized that the
mechanism by which PTKs contribute to late PC is the
modulation of iNOS activity. Accordingly, the present study
had three aims. First, we determined whether administration
of the PTK inhibitor LD-A before the first ischemic stress (on
day 1) blocks the development of late PC against myocardial
stunning. Second, we investigated whether administration of
LD-A before the second ischemic stress (on day 2) abrogates
the cardioprotection afforded by late PC against stunning.
Finally, we assessed whether LD-A (given on day 1 or day 2)
interferes with the increase in iNOS activity that underlies the
cardioprotection afforded by late PC. All studies were performed in conscious rabbits. The rationale for using a
conscious animal model was to obviate potential problems
resulting from factors associated with open-chest preparations, such as the exaggerated generation of reactive oxygen
species after myocardial ischemia/reperfusion,31 which could
have a major impact on the severity of myocardial stunning,31,32 and the trauma of a thoracotomy and the ensuing
inflammatory reaction, which may lead to release of cytokines. LD-A was chosen because (1) this agent is more
selective for PTKs than other inhibitors33,34 and (2) it has
previously been shown to inhibit Src and Lck PTKs in our
conscious rabbit model of late PC,35 which enabled us to
assess the functional significance of Src and Lck PTK
activation in this cardioprotective phenomenon.
Materials and Methods
Experimental Protocol
Phase A: Studies of Myocardial Stunning
The experimental protocol consisted of 3 consecutive days (days 1,
2, and 3, respectively) of coronary artery occlusions (a sequence of
six 4-minute coronary occlusion/4-minute reperfusion cycles) (Figure 1). Rabbits were assigned to 3 groups (Figure 1): group I
(control) did not receive any treatment; groups II and III received an
intravenous bolus of LD-A (1 mg/kg) 10 minutes before the first
coronary occlusion on day 1 or day 2, respectively. Regional
myocardial function was assessed as systolic thickening fraction, as
previously described.4,5,36 At the conclusion of the study, the size of
the occluded/reperfused coronary vascular bed was determined by
postmortem perfusion.4,5
Phase B: Studies of NOS Activity
Rabbits were assigned to 7 groups: group IV (sham control), group
V (PC-day 2), group VI (PC-day 21LD-A on day 1), group VII
(PC-day 21LD-A on day 2), group VIII (PC-day 3), group IX
(PC-day 31LD-A on day 2), and group X (LD-A without PC)
(Figure 1). Rabbits in group IV did not receive coronary occlusion/
reperfusion and were euthanized a minimum of 10 days after
surgery. Rabbits in groups V, VI, and VII underwent a sequence of
six 4-minute occlusion/4-minute reperfusion cycles as in phase A; in
groups VI and VII, LD-A (1 mg/kg IV) was administered 10 minutes
before the first coronary occlusion on day 1 and day 2, respectively.
Rabbits in groups V and VI were euthanized 24 hours later. Rabbits
in group VII were euthanized 25 minutes after the administration of
LD-A (time interval corresponding to the interval elapsed between
the administration of LD-A and the mid-part of the 6 occlusion/
reperfusion cycles in group III). Rabbits in groups VIII and IX
underwent a sequence of six 4-minute occlusion/4-minute reperfusion cycles on days 1 and 2 and were euthanized 24 hours later; in
group IX, LD-A was given on day 2 as in group III (1 mg/kg IV 10
minutes before the first coronary occlusion). Rabbits in group X
received the same dose of LD-A (1 mg/kg IV) and were euthanized
25 minutes later (similar to group VII). In all 7 groups, the heart was
1156
Circulation Research
December 3/17, 1999
citrulline using a modification of the procedure of Bredt and
Snyder.37
Measurement of Nitrite and Nitrate
Tissue nitrite was assayed by using the Griess reaction as modified
by Gilliam et al.38 Tissue nitrate was determined after conversion of
nitrate to nitrite with Aspergillus nitrate reductase.38 All assays were
performed in duplicate.
Statistical Analysis
Data are reported as mean6SEM. One-way or two-way repeatedmeasures ANOVA followed by Student’s t tests for paired or
unpaired data with the Bonferroni correction was used, as
appropriate.39,40
An expanded Materials and Methods section is available online at
http://www.circresaha.org.
Results
A total of 63 conscious rabbits were used in the present study
(2 for the pilot studies, 23 for phase A, and 38 for phase B).
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Pilot Studies
Figure 1. Experimental protocol. In phase A (studies of myocardial stunning), 3 groups of rabbits were studied. All groups
underwent a sequence of six 4-minute coronary occlusion (O)/4minute reperfusion (R) cycles followed by a 5-hour observation
period on 3 consecutive days (days 1, 2, and 3). Rabbits in
group I (n57, control) received no treatment. On day 1, rabbits
in group II (n57, LD-A on day 1) received an intravenous bolus
of LD-A (1 mg/kg) 10 minutes before the first coronary occlusion. Rabbits in group III (n57, LD-A on day 2) received the
same dose of LD-A 10 minutes before the first coronary occlusion on day 2. In phase B (studies of NOS activity), 7 groups of
rabbits were studied. Rabbits in group IV (n55, sham) underwent surgical instrumentation but did not receive O/R; the rabbits were euthanized a minimum of 10 days after surgery.
Groups V, VI, and VII underwent 6 cycles of 4-minute O/4minute R on day 1. Rabbits in group V (n55, PC-day 2) received
no treatment and were euthanized 24 hours later. Rabbits in
group VI (n55, PC-day 21LD-A on day 1) received LD-A (1
mg/kg IV) 10 minutes before the first coronary occlusion on day
1 and were euthanized 24 hours later. Rabbits in group VII
(n56, PC-day 21LD-A on day 2) were euthanized 25 minutes
after receiving the same dose of LD-A (1 mg/kg IV) on day 2.
Rabbits in groups VIII (PC-day 3) and IX (PC-day 31LD-A on
day 2) underwent a sequence of six 4-minute O/4-minute R
cycles on days 1 and 2 and were euthanized 24 hours later; in
group IX, LD-A was given on day 2 as in group III (1 mg/kg IV
10 minutes before the first coronary occlusion). Rabbits in group
X (n55, LD-A without PC) received the same dose of LD-A (1
mg/kg IV) without undergoing ischemic PC and were euthanized
25 minutes later.
excised, and myocardial samples ('0.5 g) were rapidly removed
from the ischemic/reperfused and the nonischemic region and frozen
in liquid nitrogen.
Measurement of NOS Activity
Tissue samples were homogenized in the appropriate buffer, and
cytosolic and membrane fractions were isolated. Ca21-dependent
(cNOS) and Ca21-independent (iNOS) NOS activities were determined by measuring the conversion of [14C]L-arginine to [14C]L-
Pilot studies were conducted in 2 rabbits to identify a dose of
LD-A that has no effect on heart rate, arterial blood pressure,
and systolic wall thickening (WTh). The concern was that
hemodynamic perturbations caused by LD-A (eg, a fall in
blood pressure or an increase in heart rate) could nonspecifically induce a late PC effect unrelated to the ischemic
stimulus. Arterial pressure was measured by cannulating the
dorsal ear artery with a 22-gauge angiocatheter under local
anesthesia (benzocaine), as previously described.4,5,12 The
IC50 of LD-A for receptor PTKs (eg, epidermal growth factor
receptor) is 0.011 mmol/L, while the IC50 for nonreceptor
PTKs (pp60c-src) is 0.5 mmol/L.33,34 We initially tested a dose
of 0.45 mg/kg IV, which was calculated to produce plasma
concentrations '5 times the IC50 of LD-A against nonreceptor PTKs. This dose did not have any hemodynamic effect
when administered on day 1 (before the PC occlusion/
reperfusion protocol) or (in another rabbit) on day 2, before
the second ischemic challenge; however, this dose failed to
block late PC against stunning. Therefore, we increased the
dose to 1 mg/kg ('10 times the IC50 for nonreceptor PTKs);
this dose did not have any significant effect on blood pressure
or heart rate but did block late PC when given on day 1 or day
2. Doses higher than 1 mg/kg would have been prohibitively
expensive. Consequently, a dose of 1 mg/kg was chosen for
the present experiments.
Phase A: Studies of Myocardial Stunning
Exclusions and Postmortem Analysis
Of the 23 rabbits instrumented for phase A, 7 were assigned
to group I (control group), 9 to group II (LD-A on day 1), and
7 to group III (LD-A on day 2). All animals assigned to the
control group completed the protocol on days 1, 2, and 3. Of
the 9 rabbits assigned to group II, 2 were excluded because of
persistent dyskinesis after the sixth reperfusion on day 1
(triphenyltetrazolium chloride staining demonstrated myocardial infarction that was most likely due to malfunction of the
occluder), and 1 died on day 3 because of ventricular
fibrillation during the fourth occlusion. Therefore, 6 rabbits in
group II completed days 1, 2, and 3, whereas 1 rabbit
Dawn et al
Tyrosine Kinases and Late Preconditioning
1157
Heart Rate During Coronary Occlusion and Reperfusion in Phase A
Heart Rate, bpm
Reperfusion
Baseline
Preocclusion
First
Occlusion
Sixth
Occlusion
30 Minutes
1 Hour
2 Hours
3 Hours
4 Hours
5 Hours
Day 1
26768
26069
261611
249614
258610
254613
240610
23765
23465
23764
Day 2
26468
26569
275612
25668
250610
24565
25266
23867
23968
23566
Day 3
25765
25765
26867
257610
259610
24869
25465
252612
24369
24068
Day 1
26169
25467
25366
25166
263616
25367
260615
255613
252616
258615
Day 2
24267
24267
253611
23467
241611
24268
237612
23068
22969
21967
Day 3
246616
246616
246614
233613
247611
23169
24069
23669
24168
235610
Day 1
241612
241612
23968
22764
21867
22367
21767
22069
21966
22568
Day 2
24369
254612
253611
242610
23467
23168
23168
231612
236612
227612
Day 3
254612
254612
251613
22969
237610
24969
227611
22763
228612
225614
Group I (control)
Group II (LD-A on day 1)
Group III (LD-A on day 2)
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Values are mean6SEM. Rabbits were subjected to a sequence of 6 cycles of 4-minute coronary occlusion/4-minute coronary reperfusion followed by a 5-hour
observation period. On day 1, rabbits in group II (LD-A on day 1, n57) received an intravenous bolus of LD-A (1 mg/kg, total volume 1 mL/kg) 10 minutes before
the first occlusion. On day 2, rabbits in group III received an intravenous bolus of LD-A (same dose and volume as on day 1 in group II) 10 minutes before the first
occlusion. Heart rate was measured before treatment (baseline), 9 minutes after treatment (preocclusion), at 3 minutes into the first coronary occlusion, at 3 minutes
into the sixth coronary occlusion, and at selected times after the sixth reperfusion.
completed only days 1 and 2. Of the 7 rabbits assigned to
group III, 1 died of ventricular fibrillation on day 3 during the
fifth occlusion. Therefore, 6 rabbits in group III completed
days 1, 2, and 3.
Postmortem analysis showed that the size of the occluded/
reperfused vascular bed was similar in the 3 groups: 0.7960.13
g (17.562.6% of left ventricular [LV] weight) in group I,
0.8360.09 g (16.161.5% of LV weight) in group II, and
0.8460.07 g (15.861.6% of LV weight) in group III. Tissue
staining with triphenyltetrazolium chloride confirmed the absence of infarction in all animals included in the final analysis. In
all rabbits, the ultrasonic crystal was found to be at least 3 mm
from the boundaries of the ischemic/reperfused region.
Regional Myocardial Function
As shown in the Table, there were no appreciable differences
in heart rate among the 3 groups, either during the sequence
of coronary occlusion/reperfusion cycles or during the 5-hour
reperfusion period. These results are in agreement with our
pilot studies and confirm that the dose of LD-A selected in
this study has no effect on hemodynamic variables in conscious rabbits. Baseline systolic thickening fraction in the
region to be rendered ischemic averaged 35.064.9%,
33.864.0%, and 34.464.2% on days 1, 2, and 3, respectively,
in group I; 41.063.2%, 40.263.8%, and 39.764.1% in group
II; and 38.563.8%, 37.863.4%, and 37.764.3% in group III
(Figures 2, 3, and 4). There were no significant differences
among the 3 groups on the same day or among different days
within the same group. In group II, thickening fraction on day
1 was 41.064.2% at baseline and 39.463.4% after administration of LD-A (preocclusion) (P5NS, Figure 3), indicating
that this agent had no significant effect on regional myocardial function. This conclusion is further corroborated by the
results in group III, in which thickening fraction on day 2 was
37.863.4% at baseline and 37.763.6% after administration
of LD-A (P5NS, Figure 4).
Group I (Control)
On day 1, thickening fraction remained significantly
(P,0.05) depressed for 3 hours after the sixth reperfusion
and recovered by 5 hours (Figure 2), indicating that the
sequence of six 4-minute occlusion/4-minute reperfusion
Figure 2. Systolic thickening fraction in the ischemic/reperfused
region in the control group (group I) at baseline, 3 minutes into
each coronary occlusion (O), 3 minutes into each reperfusion
(R), and at selected times during the 5-hour reperfusion interval
after the sixth occlusion. Measurements taken on day 1 are represented by the dashed line with open circles, measurements
taken on day 2 are represented by the continuous line with solid
circles, and measurements taken on day 3 are represented by
the interrupted line with solid triangles (n57 for all 3 days).
Thickening fraction is expressed as a percentage of baseline
values. Data are mean6SEM.
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December 3/17, 1999
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Figure 3. Systolic thickening fraction in the ischemic/reperfused
region in group II (LD-A on day 1) before administration of LD-A
(baseline), 9 minutes after LD-A (immediately before the first
occlusion, preocclusion [Pre-O]), 3 minutes into each coronary
occlusion (O), 3 minutes into each reperfusion (R), and at
selected times during the 5-hour reperfusion interval after the
sixth occlusion. Measurements taken on day 1 are represented
by the dashed line with open circles (n57), measurements taken
on day 2 are represented by the continuous line with solid circles (n57), and measurements taken on day 3 are represented
by the interrupted line with solid triangles (n56). To facilitate
comparisons, the data pertaining to day 1 of group I (control
group) are also shown (thick interrupted line without symbols,
n57). Thickening fraction is expressed as a percentage of Pre-O
values. Data are mean6SEM.
cycles resulted in severe myocardial stunning that lasted, on
average, 4 hours. On days 2 and 3, however, the recovery of
WTh was markedly improved after the 6 occlusion/reperfusion cycles compared with day 1 (Figure 2). The total deficit
of WTh after the sixth reperfusion was 52% and 53% less on
days 2 and 3, respectively, compared with day 1 (P,0.01)
(Figure 5). Thus, as expected,4,5,12,41 myocardial stunning was
attenuated markedly, and to a similar extent, on days 2 and 3
compared with day 1.
Group II (LD-A on Day 1)
On day 1, the recovery of WTh (Figure 3) and the total deficit
of WTh (Figure 5) were similar to values observed in the
Figure 4. Systolic thickening fraction in the ischemic/reperfused
region in group III (LD-A on day 2) at baseline, 9 minutes after
LD-A (immediately before the first occlusion, Pre-O), 3 minutes
into each coronary occlusion (O), 3 minutes into each reperfusion (R), and at selected times during the 5-hour reperfusion
interval after the sixth occlusion. Measurements taken on day 1
are represented by the dashed line with open circles (n57),
measurements taken on day 2 are represented by the dashed
line with solid circles (n57), and measurements taken on day 3
are represented by the interrupted line with solid triangles (n56).
To facilitate comparisons, the data pertaining to day 1 of group
I (control group) are also shown (thick interrupted line without
symbols, n57). Thickening fraction is expressed as a percentage of Pre-O values. Data are mean6SEM.
control group, indicating that LD-A had no appreciable effect
on the severity of myocardial stunning in nonpreconditioned
myocardium. On day 2, however, the recovery of WTh during
the 5-hour period after the sixth reperfusion was not improved compared with day 1 (Figure 3), and the total deficit
of WTh on day 2 was not significantly different from that
observed on day 1 (Figure 5). The total deficit of WTh on day
2 was 90% greater than the corresponding value in control
rabbits (P,0.01) and similar to that observed in control
rabbits on day 1 (Figure 5). On day 3, the recovery of WTh
in LD-A–treated rabbits was markedly improved compared
with day 2 (Figure 3) and was similar to that noted on day 2
Figure 5. Total deficit of WTh after the sixth
reperfusion on days 1, 2, and 3 in groups I, II,
and III (control [n57], LD-A on day 1 [n57], and
LD-A on day 2 [n57] groups, respectively). In
groups II and III, only 6 of the 7 rabbits were
studied on day 3 (see text). The values of total
deficit of WTh in individual rabbits are illustrated
in the left panel; the mean6SEM values of total
deficit of WTh are depicted in the right panel.
The total deficit of WTh was measured in arbitrary units, as described in the text.
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Tyrosine Kinases and Late Preconditioning
1159
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Figure 6. Ca21-independent NOS (iNOS) activity
in the homogenate (cytosolic1membranous fractions) of myocardial samples obtained from rabbits that underwent surgical instrumentation but
did not receive coronary occlusion/reperfusion
(sham controls [group IV]), from rabbits subjected
24 hours earlier (day 1) to six 4-minute coronary
occlusion/reperfusion cycles (PC-day 2 [group
V]), from rabbits subjected 24 hours earlier to six
4-minute coronary occlusion/reperfusion cycles
after pretreatment with 1 mg/kg IV LD-A (PC-day
21LD-A on day 1 [group VI]), from rabbits subjected 24 hours earlier (day 1) to six 4-minute
occlusion/reperfusion cycles and given the same
dose of LD-A on the second day 25 minutes
before euthanasia (PC-day 21LD-A on day 2
[group VII]), from rabbits subjected to six
4-minute occlusion/reperfusion cycles on day 1
and on day 2 and euthanized on day 3 (PC-day 3
[group VIII]), from rabbits subjected to six
4-minute occlusion/reperfusion cycles on day 1
and on day 2, given LD-A on day 2 (same dose
as group VII), and euthanized on day 3 (PC-day
31LD-A on day 2 [group IX]), and from rabbits
subjected to surgical instrumentation but not to coronary occlusion/reperfusion and given the same dose of LD-A 25 minutes before
euthanasia (LD-A without PC [group X]). iNOS activity was measured as L-NAME–inhibitable L-citrulline production in the absence of
Ca21, as described in Materials and Methods. Ischemic PC resulted in a robust increase in iNOS activity in the ischemic/reperfused
region on day 2, which was suppressed by administration of LD-A either on day 1 or on day 2. Administration of LD-A on day 2 had no
effect on the increase in iNOS activity on day 3. Data are mean6SEM.
in the control group (Figure 2). The total deficit of WTh was
66% less than that noted on day 2 in the same animals
(P,0.01) and was comparable to that noted on day 2 in
control rabbits (Figure 5). Thus, the sequence of 6 coronary
occlusions and reperfusions performed on day 1 after the
administration of LD-A failed to induce late PC against
stunning on day 2, but the same sequence performed on day
2 did precondition against stunning on day 3.
Group III (LD-A on Day 2)
On day 1, the recovery of regional function was similar to that
observed in the control group (Figures 4 and 5). On day 2,
however, both the recovery of WTh (Figure 4) and the total
deficit of WTh (Figure 5) were similar to values observed on
day 1. The total deficit of WTh on day 2 was 86% greater
than the corresponding value in control rabbits (P,0.01) and
was similar to that observed in control rabbits on day 1
(Figure 5). Thus, administration of LD-A on day 2 completely abrogated the protective effects of late PC. On day 3,
the recovery of WTh was markedly improved compared with
day 2 (Figure 4) and was similar to that observed on day 3 in
the control group (Figure 2). The total deficit of WTh was
59% less than that noted on day 2 in the same animals
(P,0.01) and was comparable to that noted on day 3 in
control rabbits (Figure 5). Thus, administration of LD-A
before the sequence of 6 coronary occlusions and reperfusions on day 2 completely abolished the late PC effect against
stunning that was induced by the ischemic stimulus on day 1.
The significant improvement in the recovery of WTh noted
on day 3 in these rabbits indicates that LD-A in itself did not
have any delayed deleterious effects on myocardial stunning.
Phase B: Studies of NOS Activity
Having observed in phase A that late PC is blocked by the
administration of LD-A on day 1 as well as on day 2, in phase
B we investigated whether PTKs participate in late PC by
modulating iNOS activity.
Exclusions
Of the 38 rabbits instrumented for phase B, 5 were assigned
to groups IV, V, VI, IX, and X, 6 to group VIII, and 7 to
group VII. One rabbit in group VII was excluded from
analysis because of probable infarction.
Effect of Ischemic PC on NOS Activity and
Myocardial Nitrite1Nitrate (NOx) Levels
As detailed in Materials and Methods, NOS activity was measured
as NG-nitro-L-arginine methyl ester (L-NAME)–inhibitable
L-citrulline production. In group V (ischemic PC-day 2), the
sequence of 6 cycles of 4-minute occlusion/4-minute reperfusion on
day 1 resulted in a robust induction of iNOS activity 24 hours later
(on day 2). In these rabbits, total (cytosolic1membranous fractions)
Ca21-independent NOS activity (iNOS activity) in the ischemic/
reperfused region was 167% higher than the activity in the nonischemic region (P,0.05) and 119% higher than the activity in the
anterior LV wall in sham control rabbits (group IV) (P,0.05)
(Figure 6). The increase in iNOS activity was more pronounced in
the cytosolic fraction (1233%, P,0.05) than in the membranous
fraction (1100%, P,0.05). No significant changes in iNOS activity were observed in the nonischemic region (Figure 6). In contrast
to iNOS activity, Ca21-dependent NOS (endothelial NOS and/or
neuronal NOS) activity in the ischemic/reperfused region did not
change appreciably 24 hours after ischemic PC (on day 2), either in
the total homogenate (cytosolic1membranous fractions) (Figure 7)
or in the individual (cytosolic and membranous) tissue fractions
(data not shown). The changes in iNOS activity were associated
with directionally concordant changes in the tissue levels of
nitrite1nitrate (NOx), the stable oxidation products of NO: as
illustrated in Figure 8, in group V total (cytosolic1membranous
fractions) myocardial NOx levels in the ischemic/reperfused region
were significantly increased 24 hours after ischemic PC (day 2)
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December 3/17, 1999
(162% versus the nonischemic region [P,0.05] and 143% versus
the anterior LV wall in group IV [sham controls] [P,0.05]).
In group VIII (ischemic PC-day 3), rabbits were subjected
to a sequence of six 4-minute occlusion/reperfusion cycles on
day 1 and to another sequence on day 2; 24 hours later (on
day 3), the increases in iNOS activity (Figure 6) and NOx
levels (Figure 8) were similar to those measured on day 2 in
group V (ischemic PC-day 2). No change in cNOS activity
was noted on day 3 (Figure 7). Thus, myocardial iNOS
activity and NOx levels were augmented both on day 2 and on
day 3, indicating sustained upregulation of iNOS by late PC.
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Effect of LD-A on NOS Activity and Myocardial NOx
In the absence of ischemic PC (group X), LD-A did not have
any appreciable effect on iNOS activity (Figure 6), cNOS
activity (Figure 7), or NOx levels (Figure 8). When LD-A was
administered before the PC ischemia on day 1 (group VI),
iNOS activity and NOx levels in the ischemic/reperfused
region 24 hours later (day 2) were not significantly different
from those measured in the nonischemic region in the same
group and in the anterior LV wall of sham control rabbits
(group IV) (Figures 6 and 8). Both iNOS activity and NOx
levels were significantly less in preconditioned rabbits treated
with LD-A on day 1 (group VI) compared with untreated
preconditioned rabbits (group V) (Figures 6 and 8). Thus,
pretreatment with LD-A completely abrogated the ischemic
PC–induced increase in iNOS activity and NOx levels observed 24 hours later (day 2) in group V.
When LD-A was administered on day 2 (group VII), iNOS
activity and NOx levels in the ischemic/reperfused region 25
minutes after LD-A did not differ significantly from those
measured in the anterior LV wall in sham control rabbits
(group IV) and were significantly less (P,0.05) than those
measured in rabbits subjected to ischemic PC (group V)
(Figures 6 and 8). Thus, administration of LD-A 24 hours
after the initial PC ischemia (day 2) markedly suppressed the
ischemic PC–induced increase in iNOS activity and abrogated the ischemic PC–induced increase in myocardial NOx
levels on day 2. In contrast, administration of LD-A on day 2
had no significant effect on the ischemic PC–induced increase in iNOS activity and NOx levels observed 24 hours
later (on day 3) (group IX).
Administration of LD-A either on day 1 or on day 2 had no
discernible effect on cNOS activity (Figure 7).
Discussion
Although previous studies have identified the initial components of the signal transduction pathway that underlies late
PC, including NO,4 –7 reactive oxygen species,9,10 and
PKC,11–14 the mechanisms downstream from these molecules
are poorly understood. Recent studies have shown that
ischemic PC activates Src and Lck PTKs,23 but the functional
significance of this observation remains unknown. In particular, no information is available regarding whether PTKs are
causally involved in late PC against myocardial stunning and,
if so, whether they participate in the development of this
adaptive phenomenon on day 1 and/or in the manifestation of
its cardioprotective effects on day 2. Furthermore, no infor-
mation is available regarding the mechanism whereby PTKs
contribute to late PC.
Salient Findings
The present investigation provides significant new insights regarding these issues. Using conscious rabbits, we found that administration of LD-A before the ischemic PC stimulus (on day 1)
completely blocked the development of protection against myocardial stunning 24 hours later, indicating that PTK activity is necessary to trigger this mechanism. The protective effects of late PC
against stunning were also abrogated when LD-A was administered
before ischemia/reperfusion on day 2, indicating that PTK activity is
also necessary to mediate this phenomenon. To our knowledge, this
is the first indication that PTKs are involved in the manifestation of
protection during the late phase of ischemic PC (on day 2). Finally,
administration of LD-A either on day 1 or on day 2 abrogated the
increase in myocardial iNOS activity and NOx levels on day 2,
indicating that the upregulation of iNOS (which mediates the
cardioprotective effects of late PC1,5,7,8) occurs via at least two
PTK-dependent pathways, one that is operative on day 1 and one
that is active on day 2.
Previous studies have implicated PTKs in early18 –21 and
late22 PC against myocardial infarction. To our knowledge,
this is the first study to demonstrate that PTKs play an
obligatory role in the development of late PC against myocardial stunning. This is also the first study (1) to identify two
distinct functions for PTKs in late PC against stunning (ie,
PTKs are required both to trigger this phenomenon on day 1
and to mediate it on day 2), (2) to directly demonstrate that
iNOS activity is upregulated during the late phase of ischemic
PC in rabbits, (3) to indicate that this upregulation of iNOS
activity requires PTK activity, and (4) to examine the effect
of PTK inhibitors on ischemic PC in conscious animals. The
finding that cardiac iNOS upregulation is PTK dependent
reveals a new signaling mechanism and a new function for
PTKs, which has significant implications for many pathophysiological processes besides ischemic PC.
Rationale for Selecting LD-A
There were several reasons for selecting LD-A instead of
genistein as a PTK inhibitor. First, the selectivity of LD-A
for PTKs versus other kinases is superior to that of
genistein. The IC50 ratio of genistein for nonreceptor PTKs
versus protein kinase A/PKC has been found to be 1:10 to
1:14,34,42 whereas LD-A is a highly selective inhibitor of
PTKs, with an IC50 of 0.011, 0.5, and .100 mmol/L for
receptor PTKs, nonreceptor PTKs, and serine-threonine
kinases (including PKC, protein kinase A, and calmodulin-dependent kinases), respectively.33,34 Thus, the IC50
ratio for nonreceptor PTKs versus PKC is ,1:200 for
LD-A34 compared with ,1:10 for genistein. The dose of
LD-A selected for the present study (1 mg/kg) was
calculated to result in peak plasma concentrations of
4.5 mmol/L, '10 times higher than the IC50 for Src PTKs
(0.5 mmol/L).33,34 Because in intact organisms LD-A is
redistributed after intravenous injection, the plasma and
tissue levels present during the 6 occlusion/reperfusion
cycles 10 to 54 minutes after the intravenous bolus of
LD-A should have been considerably lower, making it very
Dawn et al
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Figure 7. Ca21-dependent NOS (cNOS [endothelial NOS
and/or neuronal NOS]) activity in the homogenate (cytosolic1
membranous fractions) of myocardial samples obtained from
rabbits that underwent surgical instrumentation but did not
receive coronary occlusion/reperfusion (sham controls [group
IV]), from rabbits subjected 24 hours earlier (day 1) to six
4-minute coronary occlusion/reperfusion cycles (PC-day 2
[group V]), from rabbits subjected 24 hours earlier to six
4-minute coronary occlusion/reperfusion cycles after pretreatment with 1 mg/kg IV LD-A (PC-day 21LD-A on day 1 [group
VI]), from rabbits subjected 24 hours earlier (day 1) to six
4-minute occlusion/reperfusion cycles and given the same
dose of LD-A on the second day 25 minutes before euthanasia (PC-day 21LD-A on day 2 [group VII]), from rabbits subjected to six 4-minute occlusion/reperfusion cycles on day 1
and on day 2 and euthanized on day 3 (PC-day 3 [group
VIII]), from rabbits subjected to six 4-minute occlusion/reperfusion cycles on day 1 and on day 2, given LD-A on day 2
(same dose as group VII), and euthanized on day 3 (PC-day
31LD-A on day 2 [group IX]), and from rabbits subjected to
surgical instrumentation but not to coronary occlusion/reperfusion and given the same dose of LD-A 25 minutes before
euthanasia (LD-A without PC [group X]). cNOS activity was
measured as L-NAME–inhibitable L-citrulline production in the
presence of Ca21 and calmodulin, as described in Materials
and Methods. Neither ischemic PC nor LD-A had any effect
on cNOS activity. Data are mean6SEM.
unlikely that the observed inhibition of late PC could be
ascribed to inhibition of PKC or other serine-threonine
kinases.
The second advantage of LD-A over genistein is that it
is much more selective for the Src family of PTKs.
Genistein and other flavonoids are general inhibitors of
PTKs with little specificity for individual enzymes.43 In
contrast, LD-A preferentially inhibits two families of
PTKs, epidermal growth factor receptor kinases and Src
PTKs.33,34 In view of the fact that previous studies in our
conscious rabbit model have shown that ischemic PC
activates Src PTKs but has no effect on epidermal growth
factor receptor PTKs,35 LD-A would appear to be a useful
tool for interrogating the Src family of PTKs. The third
reason for selecting LD-A was that this agent (at the same
dose used in the present study) has been documented to
block Src PTK activation during ischemic PC.35 In view of
these facts, the actions of LD-A documented in the present
study suggest a critical role of the Src family of PTKs both
in the development of late PC on day 1 and in the
manifestation of its cardioprotective effects on day 2.
Tyrosine Kinases and Late Preconditioning
1161
Figure 8. Total (cytosolic1membranous fractions) myocardial content of NOx in myocardial samples obtained from rabbits that
underwent surgical instrumentation but did not receive coronary
occlusion/reperfusion (sham controls [group IV]), from rabbits subjected 24 hours earlier (day 1) to six 4-minute coronary occlusion/
reperfusion cycles (PC-day 2 [group V]), from rabbits subjected 24
hours earlier to six 4-minute coronary occlusion/reperfusion cycles
after pretreatment with 1 mg/kg IV LD-A (PC-day 21LD-A on day
1 [group VI]), from rabbits subjected 24 hours earlier (day 1) to six
4-minute occlusion/reperfusion cycles and given the same dose of
LD-A on the second day 25 minutes before euthanasia (PC-day
21LD-A on day 2 [group VII]), from rabbits subjected to six
4-minute occlusion/reperfusion cycles on day 1 and on day 2 and
euthanized on day 3 (PC-day 3 [group VIII]), from rabbits subjected
to six 4-minute occlusion/reperfusion cycles on day 1 and on day
2, given LD-A on day 2 (same dose as group VII), and euthanized
on day 3 (PC-day 31LD-A on day 2 [group IX]), and from rabbits
subjected to surgical instrumentation but not to coronary occlusion/reperfusion and given the same dose of LD-A 25 minutes
before euthanasia (LD-A without PC [group X]). Nitrite was measured by using the Griess reaction; nitrate was determined after
conversion to nitrite as described in Materials and Methods. Ischemic PC resulted in a significant increase in myocardial NOx levels,
which was abrogated when LD-A was administered before ischemic PC (day 1) or 24 hours later (day 2). Administration of LD-A on
day 2 had no effect on the increase in NOx levels on day 3. Data
are mean6SEM.
Role of PTKs in the Development of Late PC
(Day 1)
Recent studies in conscious rabbits have identified a pivotal role
of the transcription regulatory protein nuclear factor (NF)-kB in
the development of late PC on day 1.41 These studies have also
found that the activation of NF-kB by ischemic PC was
prevented by pretreatment with LD-A (given at the same dose
that was used in the present investigation), indicating that the
recruitment of this transcription factor requires PTK signaling.41
However, the mechanism whereby PTK-dependent recruitment
of NF-kB produces delayed cardioprotection remained unknown. The present findings that pretreatment with LD-A on
day 1 abrogated the increase in iNOS activity and tissue NOx
levels 24 hours later (Figures 6 and 8) indicate that PTK
signaling on day 1 is required for the upregulation of iNOS on
day 2. NF-kB is known to be a central mechanism controlling
iNOS induction,44,45 and mounting evidence indicates that the
late phase of PC is mediated by iNOS activity.5,7,8 Thus, based
on the fact that the mobilization of PTKs shortly after the initial
PC stimulus (on day 1) is essential not only for the activation of
NF-kB, as shown previously,41 but also for the increase in iNOS
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activity and for the acquisition of cardioprotection 24 hours later,
as shown in the present study (Figures 5, 6, and 8), we propose
that PTKs participate in the genesis of late PC by promoting
iNOS transcription via an NF-kB–dependent pathway.
This paradigm is consistent with emerging evidence in noncardiac cells, in which tyrosine phosphorylation has been found
to be necessary for NF-kB mobilization. For example, the PTK
inhibitors herbimycin A and genistein can block hypoxiainduced phosphorylation of inhibitory kBa (IkBa) on tyrosine
residues and the consequent activation of NF-kB in Jurkat T
cells.46 Recently, it has been shown that tyrosine phosphorylation of IkBa induced by the phosphatase inhibitor pervanadate
promotes NF-kB DNA-binding activity without degradation of
IkBa.47 In that study, a PTK of the Src family (Lck) was proven
essential for pervanadate-induced IkBa tyrosine phosphorylation and NF-kB activation.47 Interestingly, Lck is activated by
ischemic PC in conscious rabbits, and the activation is blocked
by LD-A, given at the same dose as in the present study,35
raising the possibility that this specific kinase may be involved in
the ischemic PC–induced recruitment of NF-kB.
In the present study, iNOS activity was augmented not only
on day 2 (group V) but also on day 3 (group VIII), demonstrating sustained upregulation of this enzyme during late PC. The
mechanism for the increased iNOS activity of day 3 appears to
be different from that on day 2, in view of the fact that the
administration of LD-A on day 2 had no significant effect on
myocardial stunning (Figures 4 and 5), iNOS activity (Figure 6),
and NOx levels (Figure 8) 24 hours later (on day 3) (group III
and IX). Thus, although the protection against stunning and the
concomitant increase in iNOS activity observed on day 2 require
PTK signaling on day 1 (groups II and VI), it appears that neither
the protection against stunning nor the concomitant upregulation
of iNOS activity observed on day 3 is dependent on PTK
signaling on day 2 (groups III and IX). These results are
congruent with the finding that, in conscious rabbits, a single
sequence of six 4-minute occlusion/reperfusion cycles elicits a
state of PC against stunning that lasts for 72 hours even when no
additional ischemic PC stimuli are applied.48 Together with this
previous study,48 the present results suggest that both the
preconditioned state and the rise in iNOS activity seen on day 3
are induced by the initial PC stimulus on day 1 and, therefore, do
not require any signaling events on day 2.
Role of PTKs in the Mediation of Late PC (Day 2)
A novel finding of the present study was that LD-A abolished
the cardioprotective effects of late PC when given on day 2,
after the preconditioned state had developed, revealing a role
of PTK signaling 24 hours after the initial ischemic stress. To
our knowledge, this is the first indication that the activity of
a cellular kinase is involved in the mediation (as opposed to
the triggering) of delayed protection after a PC stimulus. This
finding impels a reassessment of current paradigms, because
it implies that the augmented iNOS activity that underlies the
late phase of PC does not result exclusively from increased
iNOS transcription49 but must also involve other mechanisms. Specifically, the fact that both the increase in myocardial iNOS activity (Figure 6) and the concomitant increase in
NOx levels (Figure 8) were abrogated by the administration of
LD-A on day 2 suggests that in addition to the synthesis of
new iNOS proteins,50 posttranslational modulation of iNOS
proteins via tyrosine phosphorylation is also critical in activating this enzyme and conferring enhanced tolerance to
myocardial ischemia/reperfusion injury. Thus, a complex
scenario emerges from the present observations, in which
PTK signaling elicited by brief ischemia upregulates iNOS
both via increased transcription (day 1) and via posttranslational modulation (day 2).
The concept that tyrosine phosphorylation is required for stressinduced iNOS proteins to be protective reveals a new function of
PTKs in cardiac pathophysiology. This concept is congruent with a
previous study51 in which tyrosine phosphorylation of iNOS in
murine macrophages was found to be associated with increased
iNOS activity. Further studies will be needed to elucidate the
mechanism whereby PTK activity is enhanced on day 2 and to
identify the specific kinase(s) involved.
Conclusions
The present study provides new insights into the role of PTKs in
cardioprotection. The results of phase A demonstrate that, in
conscious rabbits, PTKs perform two distinct functions within
the signal transduction pathway that underlies late PC against
myocardial stunning: on day 1, they are essential for the
development of the late PC effect, whereas on day 2, they are
essential for its manifestation. The results of phase B are the first
direct evidence that iNOS activity is enhanced during late PC in
rabbits. Furthermore, phase B provides a mechanistic basis for
the results of phase A, in that it indicates that PTKs are involved
in the modulation of cardioprotective iNOS activity at two
distinct levels: at an early stage on day 1 and at a late stage on
day 2. This heretofore unrecognized bifunctional role of PTKs
suggests a new pathophysiological paradigm for the late phase of
ischemic PC and has broad implications for our understanding of
the signaling mechanisms that underlie the response of the heart
to ischemic stress and, possibly, other stresses. The present
findings support the concept that tyrosine phosphorylation
serves as a critical regulatory mechanism for stress-activated
gene upregulation in the heart.
Acknowledgments
This study was supported in part by National Institutes of Health
grants R01 HL-43151 and HL-55757 (Dr Bolli), by Kentucky
American Heart Association Fellowships KY-96-F-24 and 9804556
(Dr Dawn) and 9804558 (Dr Takano), and by the Jewish Hospital
Research Foundation, Louisville, Ky. We gratefully acknowledge
Gregg Shirk and Wen-Jian Wu for expert technical assistance and
Trudy Keith for expert secretarial assistance.
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Bifunctional Role of Protein Tyrosine Kinases in Late Preconditioning Against Myocardial
Stunning in Conscious Rabbits
Buddhadeb Dawn, Yu-Ting Xuan, Yumin Qiu, Hitoshi Takano, Xian-Liang Tang, Peipei Ping,
Supratim Banerjee, Michael Hill and Roberto Bolli
Circ Res. 1999;85:1154-1163
doi: 10.1161/01.RES.85.12.1154
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1999 American Heart Association, Inc. All rights reserved.
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